Rapid and visual detection of Tacheng tick virus 1 using loop-mediated isothermal amplification technique

Tacheng tick virus 1 (TcTV-1) is an emerging tick-borne nairovirus associated with human febrile illness. To date, no reliable detection method for TcTV-1 has been established. In this study, we developed and evaluated a rapid loop-mediated isothermal amplification (LAMP) assay for the detection of TcTV-1.

The primers were designed based on the nucleocapsid protein (NP) gene of TcTV-1. Sensitivity was assessed using ten-fold serial dilutions of recombinant plasmids containing the target sequence. Specificity was evaluated using cDNA from Songling virus (SGLV), Yezo virus (YEZV), Tick-borne encephalitis virus (TBEV), Severe fever with thrombocytopenia syndrome virus (SFTSV), and Beiji nairovirus (BJNV). The assay was validated using field-collected tick samples.

The TcTV-1-specific LAMP assay detected as few as 1×10^-1 copies/μL within 60 minutes at 65 °C and specifically amplified TcTV-1, with no cross-reactivity to SGLV, YEZV, TBEV, SFTSV, or BJNV. Positive reactions exhibited a clear color change from purple to blue, indicating a robust colorimetric response. A total of eight tick specimens (16.0%; 95% CI: 7.2-29.1) tested positive for TcTV-1 using both the established LAMP assay and SYBR Green real time quantitative polymerase chain reaction (RT-qPCR), demonstrating 100% sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the LAMP assay.

We report a TcTV-1-specific LAMP assay with high sensitivity, specificity, and cost-effectiveness, making it a practical tool for use in field-based or resource-limited settings.